Reaction site array, preparation process of it, reaction process using it and quantitative determination method of substance in sample solution using it

ABSTRACT

A reaction site array to be used in conducting a plurality of reactions between two or more kinds of substances in a liquid medium with a trace amount is provided. The reaction site array comprises a plurality of reaction sites separated each other, each of the reaction sites is composed of a first region and a second region, the second region is raised from the first region to separate the first regions each other, and the first region has an affinity to the liquid medium and the second region has an affinity to the liquid medium lower than that of the first region. A preparation process of the reaction site and a reaction process using the reaction site array are also provided.

BACKGROUND OF THE INVENTION

[0001]1. Field of the Invention

[0002] The present invention relates to a reaction site array havingplural reaction sites, the preparation process thereof, the reactionprocess using the reaction site array and a quantitative determinationmethod of a substance in a sample solution using the reaction sitearray, for the use of screening of chemicals such as curative medicines,of gene fingerprinting, of gene sequencing by hybridization(SBH:Sequencing By Hybridization) and of simultaneous multi-detection ofsubject materials, which can be used for so-called combinatorialchemistry where plural reactions are carried out in a trace amount atthe same time.

[0003] 2. Related Background Art

[0004] Recently, so-called combinatorial chemistry has been attractingattentions, in which, for example, a plurality of oligopeptides expectedto interact with the aimed medicine are prepared, and the interactionbetween the oligopeptides and the various chemicals to be screened areanalyzed, to identify the aimed medicine. Because the approach of randomdrug design is inefficient, and the evaluation of the designed andsynthesized drugs with animal tests etc. is time consuming andexpensive, combinatorial chemistry is now required as an alternativemeasure.

[0005] As probes for such a combinatorial chemistry, there areabove-mentioned oligopeptides. As a means to bind such probes onto thesolid, latex particles having functional groups on their surfaces tobind the probe can be commercially available (Calbiochem-NovobiochemInc.). Further, U.S. Pat. No. 5,143,854 discloses a preparation methodof an oligopeptide array using photolytic protecting groups and aphotolithography process in combination.

[0006] In detecting target nucleic acids having a certain base sequenceby using a nucleic acid probe, instead of conventional methods such asSouthern hybridization, a method has been proposed where plural types ofnucleic acid probes are immobilized onto a solid support and then testsamples including target nucleic acids are hybridized thereto and thedetection is conducted as in combinatorial chemistry.

[0007] For example, Japanese National Publication of PCT Application No.3-505157 discloses an analytical device for polynucleotide sequencewhich comprises the entire or specific parts of a full set ofoligonucleotides with a certain length immobilized onto a support.Further, in U.S. Pat. No. 5,202,231, a similar analytical method forsequencing by hybridization of polynucleotide is proposed. In U.S. Pat.No. 5,424,186, a preparation process of a nucleic acid probe array ontoa solid support by a combination use of photolytic protecting groups anda photolithography process is disclosed.

[0008] In enzyme-immunoassay, generally, reaction is carried out on amicroplate having maximum 96 wells and the results are read by amicroplate reader for simultaneous multi-item or multi-sample reactionand detection. This method has a limitation in high throughput analysisof a large number of samples.

[0009] One of the main concerns of combinatorial chemistry is how tosupply various reaction species to a reaction site effectively, in otherwords, how to supply effectively a variety of reaction species each in asmall amount (in a small liquid volume) to a reaction site without crosscontamination. From this viewpoint, the microplate method describedabove has theoretical limitations, although efficient and throughputsystems have been developed recently using robot technology. It hasanother problem that a relatively large volume of liquid, i.e., fromabout 20 μl to about 100 μl, is necessary to be supplied to one well.

[0010] Also in the synthetic method of a probe array on a solid phaseusing a photolytic protection group and photolithography described inthe above-mentioned U.S. Pat. Nos. 5,143,854 and 5,424,186, although itis possible to array a variety of probes onto a support, each probe lieson the same plane so that substances to be reacted with the probe aresupplied to the entire probes, making it impossible to conduct differentreactions with each probe. In addition, as the probes, oligopeptides oroligonucleotides which are synthesized on a solid support are usedwithout any purifiying treatment, thus it is not possible to confirmwhether desired probes are synthesized, and by-products which areinevitably synthesized during the probe synthesizing steps such asoligomers shorter than the probes etc. cannot be removed.

[0011] As a means to solve these problems, there has been proposed tosupply reaction species already synthesized and purified to the reactionsite using a microdispenser. For example, Khrapko et al. introduce amethod to make a DNA probe array by spotting a DNA solution using amicropipet onto a polyacrylamide gel (DNA Sequencing 1, 675-388, 1991).According to this method, a DNA solution of a relatively small amountcan be fed, but the region to which the DNA solution be fed cannot bespecified, thus causing a problem in quantitation. Also, crosscontamination between the juxtaposed spots may occur when probesolutions are fed. The same problem may arise when the other reactionspecies is fed.

[0012] There has been also proposed a method for stepwise synthesis ofnucleic acid probes performed on mainly porous solid matter, where theink-jet method is employed to reduce the feeding amounts of reactionspecies and to achieve reactions in various kind and a large number(International Publication of PCT Applicatin No. WO95/25116). Thismethod has problems in stepwise synthesis of probes and in thenot-specified feeding regions.

[0013] There have been proposed some measures to solve the problem thatthe regions for the reaction species can not be specified.

[0014] Chrisey et al., for example, introduce a method where a silanecoupling agent having appropriate functional groups is applied onto asupport and subjected to patterning, then onto which DNA probe solutionsare spotted to prepare a DNA probe array (Nucleic Acid Research, Vol.24,Number 15, 3040-3047, 1996).

[0015] Lemmo et al. introduce a method to feed reagents using amicrodispenser into each well of a polypropyrene sheet (plate) molded tohave a large number of wells on its surface (Anal. Chem., 69, 543-551,1997), specifically, a reagent solution of about 8 μl each is fed with amicrodispenser into each well of a polypropyrene resin plate having48×48 wells. The well size is presumed to be 3 mm in diameter and 2 mmin depth, and the size of the molded plate is described to be 8.5inches×11 inches. With molding, the feasible well size is about severalmillimeters as in the above mentioned case, and when the whole platesize is considered, the number of wells composing the array is not morethan 48×48, and the entire plate size is not so small. When actuallyused in combinatorial chemistry, much more kinds of probe species aredesirably used and a much smaller plate is desirable. In addition, sincea plate made of polypropylene is water repellent, it is difficult todistribute aqueous solutions of biomaterials such as nucleic acid intosmall wells, and undesirable cross contamination may occur between theadjacent wells.

[0016] Japanese National Publication of PCT Application No. 7-508831discloses a method to supply a nucleic acid probe solution using amicrodispenser to patterned regions of a silicone support. According tothis method, both the probe species number and array size seem to fillthe requirement, but there still remains the problem of crosscontamination when probes are fed or when test samples are applied.

[0017] According to the method disclosed in International Publication ofPCT Application No. WO95/35505, a nitrocellulose filter backed with anon water-permeable film is sectioned with silicone rubber and then tothese sections a DNA solution is supplied to form a DNA array bynon-covalent bonding. There disclosed is a method to examine pluralsamples at the same time without cross contamination by providing pluralsets of sections divided with silicone rubber on the support, but notspecifically about individual DNA reaction regions.

SUMMARY OF THE INVENTION

[0018] According to one aspect of the present invention, there providedis a process for producing a reaction site array which comprises aplurality of reaction sites to conduct a reaction between two or morekinds of substances in a liquid medium, each of the reaction sites beingcomposed of a first region having a first affinity to the liquid mediumand separated from each other by a second region having a secondaffinity to the liquid medium which is lower than the first affinity,and the second region being raised from the first region, the processcomprising the steps of:

[0019] providing a support; and

[0020] forming a matrix pattern having the second affinity and raisedfrom the support surface, to form the first region composed of thesupport surface exposed corresponding to the matrix pattern and thesecond region composed of the matrix pattern.

[0021] According to another aspect of the present invention, thereprovided is a reaction site array comprising a plurality of reactionsites to conduct a reaction between two or more kinds of substances in aliquid medium, wherein each of the reaction sites is composed of a firstregion having a first affinity to the liquid medium and separated fromeach other by a second region having a second affinity to the liquidmedium which is lower than the first affinity, and the second region israised from the first region.

[0022] According to the further aspect of the present invention, thereprovided is a process for conducting a reaction between two or morekinds of substances in a liquid medium comprising the steps of:

[0023] providing a reaction site array comprising a plurality ofreaction sites being composed of a first region having a first affinityto the liquid medium and separated from each other by a second regionhaving a second affinity to the liquid medium which is lower than thefirst affinity, and the second region being raised from the firstregion, and

[0024] applying the substances to at least one of the reaction sites andreacting the substances in the sites.

[0025] According to the further aspect of the present invention, thereprovided is a process for quantifying a first substance contained in asample liquid comprising the steps of:

[0026] a) providing a reaction site array comprising a plurality ofreaction sites, each of the reaction sites being composed of a firstregion having a first affinity to the sample liquid and separated fromeach other by a second region having a second affinity to the sampleliquid which is lower than the first affinity, and the second regionbeing raised from the first region;

[0027] b) supplying the sample liquid to the reaction site;

[0028] c) supplying to the reaction site a reagent providing adetectable and quantifiable signal when interacting with the firstsubstance to enable the quantitative detection of the first substance;and

[0029] d) quantitatively detecting the signal.

[0030] According to the present invention, the reaction sites are wellsformed on a substrate in a matrix-like pattern, and the bottom (thefirst region) of the well is the exposed substrate having a highaffinity to the liquid medium and the surrounding wall (the secondregion) raised from the substrate is made of a material having a lowaffinity to the liquid medium. Such a constitution enables smoothfeeding of the reaction solution comprised of the liquid medium andreaction substances, prevention of the solution from flowing over theraised region because of the low affinity of the surface of the raisedpart to the solution, that is, prevention of cross contamination betweenthe adjacent wells. Due to smooth feeding of the solution to the wells,the solution can be fed almost several ten times as much as the volumeof the well.

[0031] According to the present invention, the reaction site arrayhaving such functions can be effectively prepared with high accuracy.

[0032] A matrix pattern forming wells can be made using a finepatterning technology described hereinafter, a large number ofsufficiently small reaction sites can be made on a chip of, for example,1 cm×1 cm.

[0033] In the present invention, that the support surface has anaffinity to a liquid medium means, it has an affinity, in addition tothe affinity to the liquid medium itself, to the liquid mediumcontaining one or more substances such as reactants, auxiliariesrequired in reaction, reagents for quantitative or qualitative analysis,and reaction products. The same is said to the “non-affinity” of theprojecting part has no affinity.

BRIEF DESCRIPTION OF THE DRAWINGS

[0034]FIGS. 1A and 1B illustrate an example of the structure of thereaction site array of the present invention; FIG. 1A is a plan view andFIG. 1B is a cross-section.

[0035]FIG. 2 is a graph which illustrates the results of quantitativedetermination of fluorescent intensity of Example 8.

[0036]FIG. 3 is a graph which illustrates the results of quantitativedetermination of fluorescent intensity of Example 9.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

[0037]FIG. 1A is a plan view of a reaction site array according to oneembodiment of the present invention and FIG. 1B is the cross-sectionthrough the line 1B-1B of FIG. 1A. This reaction site array has aplurality of well 3 as reaction sites arrayed onto substrate 1 in amatrix form. The wells 3 are separated each other by projecting matrixpattern 2 raised from first region 4 of the well 3. The first region 4is consisted of the exposed surface of the support 1, and the surface ofthe support 1 has an affinity to the liquid medium which is used for thereaction of, for example, two kinds of substances. On the other hand,the surface of the projecting matrix pattern 2 (the second region) has alow affinity to the liquid medium compared to the first region 4.

[0038] Specifically, when the liquid medium of the reaction system is anaqueous medium (water, or a liquid medium mainly containing water), itis preferable that the surface of the support 1 is hydrophilic and thesurface of the projecting matrix pattern 2 hydrophobic. Contrary, whenthe liquid medium is not aqueous, it is preferable that the surface ofthe support 1 is lipophilic and the surface of the projecting matrixpattern 2 non-lipophilic.

[0039] More specifically, when the liquid medium is an aqueous one, thesupport 1 can be made of glass, metal or silicone wafer; glass, metal,silicone wafer, resin, or resin film those treated to have a hydrophilicsurface; or glass, metal, silicone wafer, resin, or resin film thosecoated with a hydrophilic layer to have a hydrophilic surface, whereasthe matrix pattern 2 can be made of a resin material to have ahydrophobic surface.

[0040] On the other hand, when the liquid medium is not aqueous, thesupport 1 can be made of a resin which can form a lipophilic surface,and the matrix pattern 2 can be made of metal, glass or the like to havea hydrophilic surface.

[0041] When the reaction is detected optically, preferably, the supportto be used is transparent or in some cases optically black. As apreferable support, it can be used a support made of glass such assynthetic quartz, fused quartz and the like; of silicone wafer; of aresin such as acrylate, polycarbonate, polystyrene, and vinyl chloride,or a support in which a black pigment or dye has been mixed. As theblack pigment, carbon black or organic black pigments can be used.

[0042] When the matrix pattern 2 is patterned by precision processing,the matrix pattern can be easily made from a photosensitive resin bylight-exposing and developing the photosensitive resin.

[0043] In the present invention, it is preferable that the affinity tothe liquid medium differs in the first region and the second region asmuch as possible. For example, when the liquid medium is an aqueoussystem, it is desirable that the substrate surface is more hydrophilicand the surface of the projected matrix pattern is more hydrophobic. Inthis case, it is possible to increase the hydrophobicity of the matrixpattern by baking the matrix pattern after light-exposure anddevelopment. Thus, this process is one of the preferable embodiments ofthe present invention. Likely, it also possible to increase thehydrophilicity of the first region by dry-etching the exposed supportsurface using the matrix pattern as a mask.

[0044] As a composing material of the matrix pattern used in the presentinvention, any material which meets the necessities of the presentinvention may be use. For example, metals such as chromium, aluminum andgold may also be used. When conducting an optical detection, the use ofblack chromium in combination with a clear support is believed to beideal, from a view point of reliability, since it is highly opaque. Butmetal has relatively high hydrophilicity, and when a metal film isformed by evaporation considering the uniformity of the film thickness,the film formed is usually several thousand Angstrom thick. Thesefeatures must be taken into consideration when a metal is utilized.

[0045] When the reaction system is an aqueous system, the first regionshould be hydrophilic. Thus, the suitable materials to form the matrixpattern are those having high hydrophobicity compared to the support andthose which can form a film about 1 μm or more thick securedly, andresin materials such as acrylate, polycarbonate, polystyrene,polyethylene, polyimide, acrylic monomer, urethane acrylate, Teflon andthe like can be preferably used.

[0046] To make the matrix pattern, for example, the substrate is coatedwith a resin, and a photoresist is applied onto the resin on thesubstrate, and after patterning the photoresist, the resin is subjectedto a patterning process such as etching. When a photosensitive resin isused, the resin itself can be patterned by a photolithographic processusing a photo mask. As such photosensitive resins, UV resists, DEEP-UVresists, UV-curing resins and the like can be used. As the UV resists,negative type resists such as cyclized polyisoprene-aromatic bisazideresists, phenol resin-aromatic azide resists, and positive type resistssuch as novolac resin-diazonaphthoquinone resists are included.

[0047] Of DEEP-UV resists, as positive type resists, there areradiolytic polymeric resists such as polymethylmethacrylate,polymethylenesulphone, polyhexalfluorobuthylmethacrylate,polymethylisopropenylketone, poly 1-trimethylsilyl propine bromide; anddissolution inhibitor resists such as O-nitrobenzyl cholate. As negativetype DEEP-UV resists, there are polyvinylphenol-3,3′diazidediphenylsulphone, glycidyl polymethacrylate and the like.

[0048] As ultraviolet-curing resins, included are polyester acrylate,epoxy acrylate and urethane acrylate containing 2 to 10% by weight ofone or more photopolymerization initiators selected from benzophenon andsubstitution derivatives thereof, benzoin and substitution derivativesthereof, acetophenone and substitution derivatives thereof, and oximecompounds such as benzyl dioxime.

[0049] As mentioned above, the material used for making the matrixpattern preferably has opaque properties to increase the sensitivity andreliability of detection when an optical detection, for example, afluorescent method is used. As such materials, there are metals, blackresins or black photosensitive resins. As the black resins or blackphotosensitive resins, the above-mentioned resins, or photosensitiveresins containing a black dye or a black pigment can be mentioned. Asthe black pigment, carbon black or black organic pigments can be used.Such a black-colored matrix pattern is called a black matrix pattern.

[0050] The shape of the first region can be selected considering theeasy formation, handling performance, and operability at detection.Although the shape can be selected from various polygons, ellipticalsand the like, a simple shape such as one illustrated in FIGS. 1A and 1Bis desirable. The array pattern of the first regions can be changedappropriately according to necessity. For example, they may be arrangedat the same intervals with the same separations as in the plan viewshown in FIGS. 1A and 1B, or they may be arranged so that those in theadjacent lines may not stand side by side.

[0051] Considering the number of reactions and the total size of arrays,the longest width of the first region separated by the matrix pattern ispreferably 300 μm or less. For example, if the plan shape of the well isa square as shown in FIGS. 1A, the side length can be 200 μm or less.When the plan shape is to be rectangular, the length of the long side ispreferably 200 μm or less, and when it to be a sphere, the diameter ispreferably 200 μm or less. The lower limit of the size can be 1 μm.

[0052] The distance between the two adjacent first regions is preferablyranging from ½ fold to 2-fold of the longest width of the first regionwhen the total size of the array, possibility of cross contamination,easy operation upon feeding of various solutions etc. are considered.The thickness of the matrix pattern (height from the support surface) ispreferably 20 μm or less, considering the making of the matrix pattern,the volume of the well, and the volume of the reaction solutions to besupplied, especially when the matrix pattern is made by aphotolithographic process. The lower limit of the thickness may be setto about 1 μm.

[0053] By setting the sizes in these ranges, a sufficient number ofreaction sites can be provided. When the supply of the solutions intothe first region for the reaction of two or more substances forcombinatorial chemistry is carried out by the ink-jet recording method,the direction of ejected ink and the volume of ejected ink in the orderof from picoliter to nanoliter as mentioned thereafter, are not alwaysthe same due to the changes in ejection conditions etc. Even in such acase, cross contamination among the adjacent first regions can beprevented by separating the first regions with projecting matrix patternhaving the height of 1 μm or more.

[0054] When making the square wells of 200 μm×200 μm×20 μm, each innervolume becomes 800 pl, and when each distance x between the adjacentwells in the structure shown in FIGS. 1A and 1B is set to be 200 μm, thedensity of the minute reaction sites becomes 625 units/cm², i.e.,reaction site densities of the order of 10² units/cm² or more fall intothe scope of the present invention. Alternatively, when the wells are 5μm×5 μm×4 μm in size and the distance between the adjacent wells is 5μm, the volume of each well becomes 0.1 pl and the reaction site densitybecomes 1,000,000 units/cm². Because the patterning with the size of 5μm×5 μm×4 μm is feasible by the today's microprocessing technology, thearrays in the order of 10⁶ units/cm² or more also fall into the scope ofthe preset invention.

[0055] Next, the reaction using the reaction site described above willbe illustrated.

[0056] In the present invention, in a certain well of the wells of thereaction site array of the structure described above, a reaction of atleast two kinds of substances in a liquid medium can be carried out. Inthese wells, the same reaction or different reactions can be carried outsimultaneously (including reactions of different reactants and differentreactant concentrations).

[0057] To construct the reaction of the reactive system in the wells,any conventional methods can be utilized. For example, when two kinds ofsubstances are reacted, a solution containing one substance is suppliedinto a well and then a solution containing the other substance is mixedtherewith to initiate the reaction. When three substances are used, themethods where these substances are added to a certain well one by one tomix together, where a solution containing two substance and anothersolution containing the remaining one are supplied into a well to mixtogether.

[0058] When the wells are formed as minute reaction sites and thevolumes of the reaction solutions are as small as from sub-picoliter tosub-nanoliter, it is preferable to take a means to prevent evaporationand/or gaseous diffusion of the fed solutions into the reaction site.For example, when the reaction system is an aqueous system, it isdesirable to place the array under the conditions of necessary constanttemperature and constant humidity.

[0059] In the present invention, the volume of the liquid supplied atthe reaction is around from 0.1 pl to 1 nl according to theabove-mentioned calculation, presuming the same amount as that of thewell is fed. In the present invention, since the matrix pattern part haslittle affinity to the liquid to be supplied, with some liquid species,it is possible to feed the extra amount of the liquid exceeding thevolume of the well, so that the liquid stays swelling by surface tensionat the well opening. In this case, for example, the solution volume offrom ten- to several ten- fold of the well volume can be supplied. Inother words, in the above-mentioned case, the liquid of from severalpicoliter to tens of nanoliter can be supplied. In any case, it is oftendifficult to supply a liquid of such a small amount with good precisionboth in terms of location and amount using a regular microdispenser ormicropipet. Thus, it is preferable to supply the reaction solutions intothe well using the ink-jet method in the present invention.

[0060] A solution for the reaction system can be fed by the ink-jetmethod, using an ink-jet head used in an ink-jet printer. In the ink-jetprinting, ink is ejected with highly precise positioning of an μm order,making it highly suitable to the supply of the reaction system into aminute reaction site array of the present invention. Since the amount ofthe ejected ink is from about 1 pl to about several nl in general, it isalso suitable to feed the reaction system solution in the presentinvention. Because the ink-jet heads are manufactured usingsemiconductor manufacturing technology, the discharging amount canfurther be adjusted to the desired volume.

[0061] Although the stepwise synthesis of a DNA probe array using suchan ink-jet method is mentioned in International Publication of PCTApplication No. WO95/25116, the substrate disclosed in it is of simpleglass or of porous glass. When a simple glass substrate is used, theregion where the applied solution spreads can not be controlled and alsothe problem of cross contamination will occur. When a porous glasssubstrate is used, the solution spreading can be controlled to someextent, but not in a quantitative way, and the problem of crosscontamination still remains. Moreover, with the inkjet method, a certainextent of fluctuation occurs in the direction of droplet ejected fromthe head, which results in the disorder of the array when a simple glassor the porous glass substrate is used. On the other hand, according tothe present invention, the spreading of the droplet can bequantitatively regulated by the projecting matrix pattern, and even ifthe fluctuation in the discharging direction occurred and the ejecteddroplet does not hit the first region precisely, the non-affinity of thematrix pattern to the ejected droplet leads the droplet into the desiredfirst region.

[0062] As the ink-jet method usable in the present invention to supply areaction system solution to the reaction site, there is the piezo-jetmethod, or the bubble-jet method utilizing thermal bubbling.

[0063] Next, the reaction species highly suitable to the reaction usingthe reaction site array of the present invention will be described. Inthe present invention, any reaction species which can react in the wellsarranged in an array may be used. But, the liquid medium of the reactionsystem must be an aqueous system, when the first region is hydrophilicand the surface of the projecting matrix pattern is hydrophobic, and theliquid medium should be an organic solvent, when the bottom of the well(first region) is lipophilic and the surface of the projecting matrixpattern is non-lipophilic. When two or more kinds of solutions are to besupplied, it is desirable that the solvents composing the solutions arecompatible.

[0064] Reaction species used in the present invention are exemplifiedby, a ligand and a receptor thereof, an oligo- or polypeptide having acertain amino acid sequence and a substance having an affinity thereto,an enzyme and a substrate thereof, an antigen and an antibody to theantigen, a nucleic acid or nucleic acid analog having a certain basesequence and a nucleic acid or nucleic acid analog having acomplementary base sequence to a certain base sequence of the formernucleic acid or nucleic acid analog. The nucleic acid or nucleic acidanalog is exemplified by DNA, RNA, or PNA. PNA is a nucleic acid analoghaving a peptide bond backbone (protein nucleic acid).

[0065] Also, an embodiment where at least one of the reaction substancesis bound to the inner surface of the well is in the scope of the presentinvention. Such reaction species are exemplified by immobilized enzymes,immobilized antibodies, immobilized nucleic acid probes, immobilizedpeptide probes and the like can be exemplified. By immobilizing at leastone reactive substances, the supply of the other reactive speciesbecomes easy and at the same time operations such as washing becomeseasy.

[0066] Next, a preparation process of the reaction site array using aclear glass substrate and a black photosensitive resin for the matrixpattern is explained. Reaction in the array thus formed is alsoexplained.

[0067] (1) A clear glass substrate is appropriately washed, dried, andthen a black photosensitive resin is applied thereto. As the applicationmethod, various methods including spin coating, die coating, and dipcoating can be used.

[0068] (2) The applied layer is subjected to interim hardening using,for example, a hot plate. And then, the layer is exposed to light usinga photo mask having a certain pattern and an exposure device of a wavelength matching to the spectral sensitivity of the photosensitive resin.

[0069] (3) Then development follows: when the photosensitive resincomposition is a negative-type, the part shielded from light with themask is washed out by the developing solution to expose the substratesurface and the light-exposed part remains as a black matrix pattern.Then the substrate is rinsed to remove the developing solution anddried.

[0070] (4) The matrix is hardened again using, for example, a hot plateto confer it the required water repellency.

[0071] (5) The substrate is subjected to dry etching using the blackmatrix as a mask to clean the engraved part in the matrix pattern to therequired cleanness.

[0072] (6) A solution (an aqueous solution in this case) of thesubstance(s) to be reacted is injected into the well at the desiredposition of the matrix pattern by the bubble-jet method.

[0073] (7) The reaction site array is placed under predeterminedreaction conditions.

[0074] (8) The necessary detection operations are carried out.

[0075] The present invention will be specifically illustrated withexamples thereinafter.

EXAMPLE 1

[0076] [Preparation of a Micro Reaction Site Array with a Black Matrix(for an Aqueous Reaction System)]

[0077] A synthetic quartz substrate was washed by sonication with anaqueous 2% sodium hydroxide solution, and treated with UV-ozone. ADEEP-UV resist containing carbon black (BK-739P, product ofShin-Nittetsu Kagaku Inc., a negative-type resist for black matrix) wasapplied by spin coating to make the film thickness 5 μm after hardening.The coated substrate was placed on a hot plate to heat at 80° C. for 5min to harden the resist.

[0078] The coated substrate was then subjected to proximity exposureusing a DEEP-UV exposure devise and a pattern mask to constrain thewidth of the black matrix pattern (corresponding to the distance betweenthe wells: x, as shown in FIGS. 1A and 1B, the same expression will beused hereinafter) to 200 μm and the size of the square wells to 200μm×200 μm, Then the black matrix pattern was formed using an inorganicalkaline aqueous solution as a developing solution and a spin developer,and then rinsed with pure water to completely remove the developingsolution. After drying using a spin dryer, the product was heated in aclean oven at 180° C. for 30 min to harden the black matrix completely.

[0079] Then, the substrate surface in each well was cleaned by plasmaashing using the black matrix as a mask. At this point, the measuredcontact angle of the black matrix surface to water was 87° indicatinglittle wettability, and the contact angle of the substrate surface towater was 22° indicating considerable wettability.

EXAMPLE 2

[0080] [Preparation of a Minute Reaction Site Array Using Black Chromium(for a Non-Aqueous Reaction System)]

[0081] An acrylic resin substrate (Deraglass, product of Asahi-KaseiKogyo) was washed by sonication with a 2% sodium hydroxide aqueoussolution and treated with UV-ozone. The resist pattern corresponding tothe first regions was formed by conventional photolithography. Thethickness of the resist was 1 μm after hardening. After the post-bakingprocess at 100° C. for 30 min, a black chromium film of 2000 angstromthick was made, and the chromium film corresponding to the first regionswas removed by the lift-off method using a resist exfoliating solution.The support was washed appropriately and after drying, the supportsurface was cleaned by plasma ashing as described in Example 1. By thisprocess, the minute reaction site array having a matrix pattern made ofblack chromium and first regions made of acrylic resin was obtained. Atthis point, the measured contact angle of the black chromium surfacewith water was 25°, indicating wettability, and the contact angle of thesubstrate surface with water was 93°, indicating little wettability.

EXAMPLE 3

[0082] [Supply of an Aqueous Solution Into a Minute Reaction Site Arrayby Ink-Jet Method-I]

[0083] A minute reaction site array of 1 cm×1 cm was made on a glasssubstrate, which was comprised of 2500 units of square first regions of100 μm×100 μm (reaction well) with the black matrix pattern of whichwidth was 100 μm, by the same manner as described in Example 1. Then, anaqueous 10 μM rhodamine B was fed into the wells in a checkered patternusing an ink-jet device, where the feed amount per well was 50 pl, equalto the volume of the reaction well. The precision of ejectionpositioning of the ink-jet devise used was ±2.5 μm. Then, an aqueoussolution of 10 μM amino-FITC was supplied in an amount of 50 pl per wellto the remaining reaction wells from another ink-jet head. Rhodamine Band amino-FITC were used because they are water soluble and theirfluorescence is suitable for observation of conditions of thedistributed liquid and cross contamination.

[0084] Using a Nikon fluorescent microscope with a G excitation filter(for rhodamine B) and a B excitation filter (for amino-FITC), theconditions of fed solutions were observed by fluorescence at themagnification of 100 times. Each solution was uniformly distributed intothe wells without forming a droplet. Moreover, the fluorescence of eachdye was found only in the wells where the dye should be found,indicating that the distribution of the solution into wells was carriedout without any cross contamination.

EXAMPLE 4

[0085] [Supply of an Aqueous Solution Into a Minute Reaction Site Arrayby Ink-Jet Method-II]

[0086] Solutions of rhodamine B and amino-FITC were distributed in thesame manner as in Example 3, except that 500 pl, 10 times as much as thewell volume, was distributed to each well. When observed using afluorescent microscope, each dye solution was supplied into each wellforming a droplet due to the water repellency of the matrix pattern. Nocross contamination was observed as in Example 3.

EXAMPLE 5

[0087] [Supply of a Non-Aqueous Solution Into a Minute Reaction SiteArray by Ink-Jet Method]

[0088] A minute reaction site array of 1 cm×1 cm was formed on anacrylic substrate, which was comprised of 2500 units of square firstregions of 100 μm×100 μm (reaction well) with the black chromium matrixpattern of which width was 100 μm, in the same manner as in Example 2.Then, a DMSO solution of 10 μM rhodamine B was supplied in the 50-foldvolume of the well volume, 50 pl per well, to every other well in acheckered pattern using an ink-jet device. Then, the same amount of aDMSO solution of 10 μM FITC was supplied into each remaining well fromanother ink-jet head. When observed using a fluorescent microscope, theDMSO solution of each dye was supplied into each well swollen from thewell (because the supplied volume was not large enough to form adroplet) due to the non-lipophilic property of the matrix pattern. Alsono cross contamination was observed as in Example 3.

EXAMPLE 6

[0089] [Feeding Model of Two Kinds of Aqueous Solutions Into a MinuteReaction Site Array by Ink-Jet Method]

[0090] In the same manner as in Example 3, the aqueous rhodamine Bsolution and the amino-FITC solution were distributed in a checkeredpattern to the wells as a model of the first reaction species, exceptthat each well received 250 pl of the solution, 5 times as much as thewell volume. Then, another 250 pl of the same solution was added to thesame well as a model of the second reaction species. When observed usinga fluorescent microscope, each solution had been distributed in eachwell in the form of a droplet due to the water repellent property of thematrix pattern, and no splash was observed in swelle of the secondfeeding. Also no cross contamination was observed.

EXAMPLE 7

[0091] [Supply of Two Kinds of Aqueous Solutions Into a Minute ReactionSite Array by Ink-Jet Method and Their Reaction]

[0092] Fifty picoliter of a TE buffer (pH 7.5) containing 40 μMsonication-fragmented salmon testes DNA was distributed into each wellas in Example 3. Then, 50 pl of a TE buffer containing 10 μM ethidiumbromide (EB) was added to the same well, and then incubated for 5 min inan incubator at 25° C. and 100% humidity, and the fluorescence wasobserved using a fluorescent microscope (with a G excitation filter).The fluorescence was observed in the wells received both solutions. Thismeans that EB and the double-stranded nucleic acid reacted in the welland the fluorescence due to the EB intercalation was observed. From thewells to which only EB was supplied (control wells), only very weakfluorescence was observed.

EXAMPLE 8

[0093] [Supply of Two Kinds of Aqueous Solutions Into a Minute ReactionSite Array by Ink-Jet Method, Their Reaction and Quantitation ofFluorescence After the Reaction-I]

[0094] DNA solutions of 0 μM, 2 μM, 5 μM, 10 μM, 20 μM, 40 μM, 100 μMand 200 μM (base pair concentration) were supplied in an amount of 50picoliter into eight wells respectively, as in Example 7. Then, 50 pl ofa 10 μM EB solution was added to each well, and incubated as in Example7. Then the fluorescent images from the fluorescent microscope were readinto an ICCD camera (Hamamatsu Photonix C2400-87) and the light volumewas quantitatively determined by an image processing devise (HamamatsuPhotonix Argus50). The signal amplification level of Argus50 wasappropriately set. The results are shown in FIG. 2. FIG. 2 indicatesthat the reaction in the minute reaction site array of the presentinvention proceeded and detected quantitatively. At a certain ratio ofDNA and EB, the fluorescence intensity reaches the saturation.

EXAMPLE 9

[0095] [Supply of Two Kinds of Aqueous Solutions Into a Minute ReactionSite Array By Ink-Jet Method, Their Reaction and QuantitativeDetermination of Fluorescence After the Reaction-II]

[0096] A 40 μM TE buffered (pH 7.5) solution of carboxyfluoreceindiacetate (Molecular Probe Inc., C369), a fluorescent substrate ofesterase, was fed by ink-jet method in an amount of 50 pl each intoseven wells of the minute reaction site array prepared in the samemanner as described in Example 3. Then, a solution of 2 units/litercholine esterase (Wako Jyunyaku) in TE buffer (pH 7.5) was added to eachwell with different time intervals so that the reaction time in theseven wells becomes 0 min, 5 min, 10 min, 15 min, 20 min, 25 min and 30min, respectively. The reaction was conducted in an incubator at 25° C.and 100% humidity. Then, after the heat treatment at 60° C. for 5 min toinactivate the enzyme, the fluorescence of carboxyfluoresceine which wasproduced by the enzyme digestion was observed and quantitated by afluorescent microscope (with a B excitation filter) +ICCD +Argus. Thesignal amplification level of Argus50 was the same as described inExample 8. The results are shown in FIG. 3. FIG. 3 indicates that thereaction in the minute reaction site array of the present inventionproceeded and detected quantitatively. The fluorescence intensityreaches the saturation due to the enzyme/substrate ratio.

EXAMPLE 10

[0097] [Preparation of a More Minute Reaction Site Array and Supply ofan Aqueous Solution by Ink-Jet Method]

[0098] A minute reaction site array of about 1 cm×1 cm was formed on aglass substrate in the same manner as in Example 1, except that theblack matrix pattern was 5 μm in width and the film thickness was 4 μm,and there contained 1,000,000 units of 5 μm×5 μm square wells. Then, asolution of 10 μM rhodamine B was fed to each well using an ink-jetdevice in a checkered pattern. The precision of ejection positioning ofthe ink-jet devise used is +0.5 μm. Then, an aqueous solution of 10 μMamino-FITC was fed in an amount of 1 pl into each of the remaining wellsfrom another inkjet head.

[0099] By using a Nikon fluorescent microscope installed with a Gexcitation filter (for rhodamine B) and a B excitation filter (foramino-FITC), and fluorescence observation was carried out at themagnification of 400 times to know the conditions of the fed solutions.The solution of each dye was found in the form of droplet as in Example4. No cross contamination was observed.

[0100] The present invention enables the preparation of a minutereaction site array suitable for the supply of the reaction species toconduct a large number of various kinds of reactions in a small region(for example, 1 cm×1 cm) on a substrate. With combined use of theink-jet method, supply of solutions of reaction species to the minutereaction site array of the present invention and their reaction, as wellas detection of the reaction and quantitative determination as neededhave been able to conducted.

What is claimed is:
 1. A process for producing a reaction site array comprising a plurality of reaction sites to conduct a reaction between two or more kinds of substances in a liquid medium, each of the reaction sites being composed of a first region having a first affinity to the liquid medium and separated from each other by a second region having a second affinity to the liquid medium which is lower than the first affinity, and the second region being raised from the first region, the process comprising the steps of: providing a support; and forming a matrix pattern having the second affinity and being raised from the support surface, to form the first region composed of the support surface exposed corresponding to the matrix pattern and the second region composed of the matrix pattern.
 2. The process according to claim 1, wherein the support surface is hydrophilic and the matrix pattern surface is hydrophobic.
 3. The process according to claim 1, wherein the support surface is lipophilic and the matrix pattern surface is non-lipophilic.
 4. The process according to claim 1 or 2, wherein the matrix pattern is made of a resin material.
 5. The process according to claim 1, wherein the matrix pattern is made by a photolithographic process.
 6. The process according to claim 5, wherein the photolithographic process comprises the steps of: making a resin layer on the support surface; forming a photoresist layer on the resin layer; irradiating light to the photoresist layer to make a pattern corresponding to the matrix pattern; developing the exposed photoresist layer; patterning the resin layer using the photoresist layer as a mask; and forming the matrix pattern by removing the photoresist layer.
 7. The process according to claim 5, wherein the photolithographic process comprises the steps of: making a photosensitive resin layer on the support surface; irradiating light to the photosensitive resin layer to make a pattern corresponding to the matrix pattern; developing the photosensitive resin layer to make the matrix pattern.
 8. The process according to claim 7, wherein the matrix pattern made of the photosensitive resin layer is further subjected to post-baking process to improve its water repellency.
 9. The process according to claim 2, wherein the support surface is hydrophilic, and the support surface composing the first region is subjected to etching using the matrix pattern as a mask to improve the hydrophilicity of the first region.
 10. The process according to claim 1, wherein the support is transparent and the matrix pattern is opaque.
 11. The process according to claim 10, wherein the matrix pattern is colored in black.
 12. The process according to claim 1, wherein the height of the matrix pattern is from 1 μm to 20 μm.
 13. The process according to claim 1, wherein a distance between the first regions adjacent each other is from ½ times to 2 times a maximum length of the first region.
 14. A reaction site array to conduct a reaction between two or more kinds of substances in a liquid medium, wherein each of the reaction sites is composed of a first region having a first affinity to the liquid medium and separated from each other by a second region having a second affinity to the liquid medium which is lower than the first affinity, and the second region is raised from the first region.
 15. The reaction site array according to claim 14, wherein a density of the reaction sites is 10² units/cm² or more.
 16. The reaction site array according to claim 14, wherein the second region is formed in a pattern form on a surface of a support, the support surface having the first affinity.
 17. The reaction site array according to claim 14 or 16, wherein the second region is composed of a resin material.
 18. The reaction site array according to claim 16, wherein the second region is made by a photolithographic process.
 19. The reaction site array according to claim 14, wherein the first region is transparent and the second region is opaque.
 20. The reaction site array according to claim 14, wherein the support is transparent and the second region is opaque.
 21. The reaction site array according to claim 20, wherein the second region is colored in black.
 22. The reaction site array according to claim 14, wherein a maximum length of the first region is 200 μm or less.
 23. The reaction site array according to claim 14, wherein a distance between the first regions adjacent each other but separated by the second region is ranging from ½ times to 2 times a maximum length of the first region.
 24. The reaction site array according to claim 14, wherein a height of the second region is from 1 μm to 20 μm.
 25. A process for conducting a reaction between two or more kinds of substances in a liquid medium comprising the steps of: providing a reaction site array comprising a plurality of reaction sites being composed of a first region having a first affinity to the liquid medium and separated from each other by a second region having a second affinity to the liquid medium which is lower than the first affinity, and the second region being raised from the first region, and applying the substances to at least one of the reaction sites and reacting the substances in the sites.
 26. The process according to claim 25, wherein the density of the reaction sites is 10² units/cm² or more.
 27. The process according to claim 25, wherein the second region is made in a pattern form on a surface of a support, the surface having the first affinity.
 28. The process according to claim 25 or 27, wherein the second region is composed of a resin material.
 29. The according to claim 25 or 27, wherein the second region is made by a photolithographic process.
 30. The process according to claim 25, wherein the first region is transparent and the second region is opaque.
 31. The process according to claim 27, wherein the support is transparent and the second region is opaque.
 32. The process according to claim 31, wherein the second region is colored in black.
 33. The process according to claim 25, wherein a maximum length of the first region is 200 μm or less.
 34. The process according to claim 25, wherein a distance between the first regions adjacent each other is ranging from ½ times to 2 times a maximum length of those first regions.
 35. The process according to claim 25, wherein a height of the second region is ranging from 1 μm to 20 μm.
 36. The process according to claim 25, wherein the two or more substances include substances relating each other as a ligand and a receptor.
 37. The process according to claim 25, wherein oligopeptides or polypeptides having a certain amino acid sequence are included in the two or more substances.
 38. The process according to claim 25, wherein proteins are included in the two or more substances.
 39. The process according to claim 38, wherein the proteins are antibodies.
 40. The process according to claim 39, wherein the proteins are antigens.
 41. The process according to claim 25, wherein enzymes are included in the two or more substances.
 42. The process according to claim 25, wherein single-stranded nucleic acids or modified nucleic acids having a certain base sequence are included in the two or more substances.
 43. The process according to claim 42, wherein the nucleic acids are oligonucleotides or polynucleotides.
 44. The process according to claim 42, wherein the nucleic acid is DNA.
 45. The process according to claim 42, wherein the nucleic acid is RNA.
 46. The process according to claim 42, wherein the modified nucleic acids is protein nucleic acid (PNA: Protein nucleic Acid).
 47. The process according to claim 25, wherein a supply of the substances to the reaction site array is conducted using ink-jet method.
 48. The process according to claim 47, wherein a supply of the substances to the reaction site array is conducted using bubble-jet method.
 49. The process according to claim 47, wherein a supply of the substances to the reaction site array is conducted using piezo-jet method.
 50. A process for quantifying a first substance contained in a sample liquid comprising the steps of: a) providing a reaction site array comprising a plurality of re action sites, each of the reaction site s being composed of a first region having a first affinity to the sample liquid and separated from each other by a second region having a second affinity to the sample liquid which is lower than the first affinity, and the second region being raised from the first region; b) supplying the sample liquid to the reaction site; c) supplying to the reaction site a reagent providing a detectable and quantifiable signal when interacting with the first substance to enable the quantitative detection of the first substance; and d) quantitatively detecting the signal.
 51. The process according to claim 50, wherein the step c) includes a process to supply to each reaction site a liquid medium containing a second substance which binds to the first substance, and the reagent, where the first region shows the first affinity to the liquid medium, and the reagent interacts with a complex of the first substance and the second substance.
 52. The process according to claim 51, wherein to each reaction site, different kinds of substances are supplied as the second substances.
 53. The process according to claim 50, wherein the density of the reaction site is 10² units/cm² or more.
 54. The process according to claim 50, wherein the second region is made in a pattern form on a surface of the support, the surface having the first affinity.
 55. The process according to claim 50 or 54, wherein the second region is composed of a resin material.
 56. The process according to any of claim 50, 54 or 55, wherein the second region is made by a photolithographic process.
 57. The process according to claim 50, wherein the first region is transparent and the second region is opaque.
 58. The process according to claim 50, wherein the support is transparent and the second region is opaque.
 59. The process according to claim 57 or 58, wherein the second region is colored in black.
 60. The process according to claim 50, wherein a maximum length of the first region is 200 μm or less.
 61. The process according to claim 50, wherein a distance between the first regions adjoined each other but separated by the second region is ranging from ½ times to 2 times of the maximum length of the first region.
 62. The process according to claim 50, wherein the height of the second region is 20 μm or less.
 63. The process according to claim 50, wherein the reagent is contained in the sample liquid.
 64. The process according to claim 51, wherein the reagent is contained in a liquid medium containing the second substance.
 65. The process according to claim 51, wherein a relation between the first substance and the second substance is a ligand and a receptor.
 66. The process according to claim 51, wherein at least one of the first substance and the second substance is an oligonucleotide or polypeptide having a certain amino acid sequence.
 67. The process according to claim 66, wherein at least one of the first substance and the second substance is a protein.
 68. The process according to claim 67, wherein the protein is an antibody.
 69. The process according to claim 67, wherein the protein is an antigen.
 70. The process according to claim 51, wherein at least one of the first substance and the second substance is an enzyme.
 71. The process according to claim 51, wherein at least one of the first substance and the second substance is a single-stranded nucleic acid or a modified nucleic acid having a certain base sequence.
 72. The process according to claim 71, wherein the nucleic acid is an oligonucleotide or a polynucleotide.
 73. The process according to claim 71, wherein the nucleic acid is a DNA.
 74. The process according to claim 71, wherein the nucleic acid is an RNA.
 75. The process according to claim 71, wherein the nucleic acid is a protein nucleic acid (PNA).
 76. The process according to claim 50, wherein supply of at least one of the sample and the reagent to the reaction site is conducted by an ink-jet method.
 77. The process according to claim 51, wherein the supply of a liquid medium containing the second substance to the reaction site is conducted by an inkjet method.
 78. The process according to claim 76 or 77, wherein the ink-jet method is a bubble-jet method.
 79. The process according to claim 76 or 77, wherein the ink-jet method is a piezo-jet method. 